Aminoacyl-tRNA synthetases (aaRS) are the enzymes ensuring faithful transmission of genetic information in all living cells. They match cognate amino acids with tRNAs, forming an aminoacyl-tRNA ester by way of an aminoacyl-adenylate intermediate. Some tRNA synthetases cannot distinguish between structurally similar amino acids with high accuracy, and thus proceed in catalysis of the noncognate reaction. However, the noncognate aminoacyl-adenylate and aminoacyl-tRNA are destroyed by additional hydrolytic activities of the enzymes (pre- and post-transfer editing or proofreading, respectively). Hydrolysis of the noncognate aminoacylated tRNA (post-transfer editing) occurs in a spatially separate editing domain. Crystal structures support a model whereby the single stranded 3'-end of the aminoacyl-tRNA is translocated from the synthetic to the hydrolytic site some 30 E distant. Hydrolysis of the noncognate aminoacyl-adenylate (pre-transfer editing) has been assumed to occur at the same remote hydrolytic site and shuttling of the intermediate was therefore proposed. However, crystal structures have never corroborated the existence of the shuttling pathway. Recently, it was shown that hydrolysis of cognate glutaminyl-adenylate, an analogous reaction to pre-transfer editing, may occur in the confines of the active site of a nonediting class I glutaminyl-tRNA synthetase. This strongly suggests that hydrolysis of noncognate aminoacyl-adenylate can occur in the homologous synthetic sites of class I editing synthetases such as isoleucyl-, valyl- and leucyl-tRNA synthetases (IleRS, ValRS and LeuRS, respectively). The long-term objective of this project is to elucidate the mechanisms of the editing activities of IleRS and ValRS toward noncognate amino acids. Two specific aims (1 and 2) are proposed to address the following questions: (i) does pre-transfer editing require tRNA and if it does what is its role? (ii) where does the hydrolysis of noncognate aminoacyl-adenylate take place?;(iii) does amino acid discrimination operate through conformational readjustment of both synthetase and tRNA during aminoacylation and/or translocation step? Addressing these questions will also require the development of suitable methodology. A new aminoacyl-adenylate synthesis assay (recently developed for the study of GlnRS), as well as novel partitioning assays (proposed herein) will be applied to follow the fate of the aminoacyl-adenylate. tRNA analogues and post-transfer editing deficient protein mutants will be used in the experiments designed to evaluate a role for tRNA and location of pre-transfer editing. Stopped flow fluorescence will be pursued, using several different fluorophores, to explore conformational readjustment of synthetase and tRNA during the course of the noncognate and cognate reactions. The data obtained will significantly improve our understanding of amino acid discrimination by editing class I aminoacyl-tRNA synthetases. PUBLIC HEALTH RELEVANCE: Accurate aminoacyl-tRNA synthesis is a prerequisite to the survival of all cells. Consequently, the proofreading activities of aminoacyl-tRNA synthetases have been conserved through evolution in spite of the high energetic cost that the reactions impose on the cell. Understanding of the editing mechanism in detail (Specific Aims 1 and 2) would allow better use of these enzymes as targets for antibacterial agents, because the editing pathway represents an additional selective target for inhibitor action.